基于ES-Cell的编程组合MYH6启动子，选择小鼠心脏起搏器细胞聚集一代
Summary
本协议描述了如何制作从小鼠多能干细胞（PSC）功能性鼻窦淋巴组织。 T-BOX3（TBX3）表达以及心肌肌球蛋白重链（MYH6）启动抗生素的选择导致了高纯度的起搏细胞聚集。这些“诱导窦房结-机构”（“iSABs”）含有80％以上的起搏细胞，显示高度增加跳动的速度，并能步伐心肌体外 。
Abstract
“病窦综合征”的治疗是根据人工心脏起搏器。这些熊的危害，如电池故障和感染。此外，它们缺乏激素响应性和总体过程是成本密集型的。从生成的PSCs“生物起搏器”有可能成为一种替代方法，还起搏细胞的胚状体的典型含量（EBS）是非常低的。所描述的协议将通过窦房结诱导TBX3与MYH6，促进基于抗生素选择“前进编程”鼠产品分成合同。这就产生心肌聚集一致> 80％生理功能起搏细胞。这些“诱导窦房结-体”（“iSABs”）被自发收缩在对应于节点的细胞从小鼠心脏中分离但未得频率（400-500 BPM），并能踱步鼠心肌体外 。使用所描述的协议的高纯度窦可以生成节点单个细胞，其例如可以用于在体外药物测试。此外，根据该协议所产生的iSABs可能成为朝向心脏组织工程的关键步骤。
Introduction
术语“病态窦房结综合征”总结了多种疾病导致的心脏起搏器系统的恶化。它包括病理，对症窦性心动过缓，窦房传导阻滞，窦性停搏以及心动过速，心动过缓综合征。由此，一个“病态窦房结综合征”常伴一般心脏疾病如缺血性心脏疾病，心肌病或心肌炎。目前，治疗方法是基于电起搏器的植入。然而，这伴随着一个数字，如感染和电池故障的风险。总体而言，并发症的发生率还是非常高的患者已经植入人工心脏起搏器。此外，相对于内源性心脏起搏器，这些设备不响应激素刺激。
未来的另一种可能依赖于哪些产品分成合同可能成为的“生物起搏器”的可用性作为合适的细胞来源，这也将是用于体外药物测试非常有价值的。然而，一个主要的问题出在胚体（EBS）中非常罕见的外观窦结细胞-这通常不超过约0.5％1。
此前，它表明“向前编程”朝特定心肌亚型是通过不同的早期心血管转录因子如中胚层特异性-后1（MesP1）和NK2转录因子相关的，轨迹5（的Nkx2.5）2的过表达是可行的， 3。对于正常尺寸和窦房结（SAN）的功能，在T-box转录因子TBX3是至关重要的，这已被证明以引发起搏器基因程序，并控制在SAN 4的分化。而这种增强的功能性起搏细胞的外观，内容依然没有整个cardiomyocytic细胞群中超过〜40％。
因此，一个额外的MYH6，促进基于抗生素选择步骤5中我们进行了介绍。这最终导致尚未未观察到心肌细胞聚集体（“诱导窦房机构;”iSABs“），其在体外表现出高度增加的跳动的频率（> 400 BPM），首次近似那些鼠心脏的和可比较的体外培养窦结细胞从鼠心脏6隔离。在异丙肾上腺素管理甚至殴打的550 BPM频率来实现。值得注意的是，iSABs包括超过80％的功能节点细胞从粗放生理得到印证分析7。最近，一些方法来使用直接重新编程19产生窦淋巴结细胞，表面标记14或药物治疗与小分子16,17进行了描述。然而，这些方法导致起搏细胞的这种高纯度和跳动频率接近鼠他艺术在iSABs观察。
此外，在已失去了他们的自发活动的跳动种植成年小鼠脑室片的体外模型中，iSABs能够集成到切片组织，因此剩余的自发主动和有力起搏心脏切片收缩7。这些iSABs的生成一个详细的协议，本文介绍。
Protocol
Representative Results
Discussion
的能力产生干细胞来源的心脏起搏细胞可允许适当心律重建中的“生物起搏器”的感觉。同样，在体外药物测试将受益于他们的可用性。的PSCs可以引起哺乳动物体包括与起搏器电池特性8,9,10,11,12,13心肌的任何细胞类型。然而，通常为“胚体”内的细胞群体是高度异质的，这不可避免地导致了可靠的选择和分离的策略的要求 – 这尤其适用于心脏结细胞的非常罕见的细胞类型。基于外源性和?…
Disclosures
The authors have nothing to disclose.
Materials
| Name of the Material/Equipment | Company | Catalog Number |
| Iscove's liquid medium with stable glutamine | Biochrom AG | FG 0465 |
| DMEM liquid medium without Na-pyruvate, with stable glutamine | Biochrom AG | FG 0435 |
| CLS type IV, CLS IV | Biochrom AG | C4-22 |
| Non-essential amino acids | Biochrom AG | K 0293 |
| FBS Superior | Biochrom AG | S 0615 |
| Sodium pyruvate (100 mM) | Biochrom AG | L 0473 |
| G 418-BC liquid (ready-to-use solution), sterile | Biochrom AG | A 2912 |
| Reagent reservoir PP f.multichannel pip. 60ml,sterile | Brand | 703409 |
| Accutase | eBioscience,Inc. | 00-4555-56 |
| Eppendorf Xplorer/Xplorer plus, electronic pipette | Eppendorf | 4861000155 |
| Falcon Cell Strainer 40µm | Falcon | 352340 |
| Penicillin/Streptomycin | GE Healthcare | P11-010 |
| Petri Dishes | Greiner BioOne | 663102 |
| DPBS without Ca and Mg | PAN-Biotech | P04-36500 |
| Fetal bovine serum | PAN-Biotech | P30-3302 |
| Leukemia inhibitory factor | Phoenix Europe GmbH | LIF-250 |
| Quadratic petri dishes | Roth | PX67.1 |
| Gelatin from cold water fish skin | SigmaAldrich | G7765 |
| 2-Mercaptoethanol | SigmaAldrich | M3148 |
| 1-Thioglycerol | SigmaAldrich | M6145 |
| Tissue culture dishes | TPP | 93100 |
| Tissue culture flask | TPP | 90076 |
| Tissue culture test plates (24 well) | TPP | 92424 |
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